Preparation method and product of propolis-ginkgo leaf complex micromolecular peptide for synergistic enhancement

Abstract

This invention belongs to the field of deep processing of plant extracts and health food technology, and discloses a preparation method for a product of propolis-ginkgo leaf compound small molecule peptide enhancement. Based on a mass ratio of 85:45 for propolis extract and ginkgo leaf extract, a three-enzyme synergistic and directional compounding system of neutral protease + cellulase + glycosidase is constructed. Enzymatic hydrolysis is performed under mild conditions of pH 6.5-7.2 and 50-55℃ for 4-6 hours. After inactivation, precision filtration and centrifugation, low-temperature vacuum concentration, and gradient spray drying, a propolis-ginkgo compound peptide powder is obtained, which is then compounded with conventional excipients to prepare the product. Without adding raw materials, changing the ingredient list, or modifying with acids, alkalis, or organic solvents, a mild and efficient conversion of active ingredients is achieved. The product has a small molecule peptide content ≥18.5%, a free flavonoid content ≥82%, a DPPH free radical scavenging rate ≥82.22% higher than traditional processes, a raw material utilization rate ≥91%, and significantly improved formulation solubility and stability.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of plant extracts and health food technology, specifically relating to a preparation method for a product of small molecule peptide enhancement of propolis-ginkgo leaf compound. Background Technology

[0002] Both propolis and ginkgo biloba L. are historically significant and proven natural active ingredients. Propolis is rich in over 300 active components, including flavonoids, phenolic acids, and terpenes, exhibiting various biological activities such as antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects. The flavonoid aglycones and terpene lactones in ginkgo biloba extract have unique effects on improving cardiovascular circulation, scavenging free radicals, and improving memory and cognition. The combined use of these two ingredients achieves a synergistic effect of antioxidant activity and improved microcirculation; therefore, propolis and ginkgo biloba compound products are widely used in the health food industry.

[0003] Currently, the mainstream production processes for propolis and ginkgo leaf compound products mainly include alcohol extraction, water extraction, or simple physical mixing methods. Taking alcohol extraction as an example, the raw materials are typically extracted with ethanol of different concentrations, concentrated under reduced pressure to obtain an extract, which is then dried, pulverized, and compounded with excipients. However, these traditional processes have the following core technical defects: First, the active ingredients exist in a large molecular bound state, resulting in extremely low bioavailability. The proteins in propolis exist as intact macromolecules, and the flavonoids in ginkgo leaf cell walls are mostly in a glycoside-bound state. The human gastrointestinal tract lacks the corresponding enzyme system to effectively break down these macromolecular proteins and glycoside-bound flavonoids into directly absorbable small peptides and free flavonoid aglycones. The bioavailability of products processed using traditional methods is only 20-30%, with a large amount of active ingredients being excreted without absorption, resulting in significant waste of efficacy.

[0004] Secondly, the product is highly lipid-soluble and poorly water-soluble, resulting in insufficient formulation stability. The active ingredients in propolis and ginkgo leaf extracts are primarily lipid-soluble, exhibiting poor dispersibility in aqueous systems. Soft capsules produced using traditional processes are prone to layering, precipitation, and clumping during their shelf life, severely impacting product uniformity, stability, and consumer experience, and shortening the product's effective shelf life.

[0005] Third, the raw material utilization rate is low, and the loss of effective components is significant. Traditional alcohol or water extraction processes have limited efficiency in extracting large molecular proteins from propolis and cell walls from ginkgo leaves. A large number of effective components are trapped in unbroken cells or undegraded protein aggregates and cannot be dissolved. The raw material utilization rate is usually only around 58%, resulting in a serious waste of raw material resources and a significant increase in production costs.

[0006] Fourth, the antioxidant effect is limited and cannot meet the demands of the high-end market. The antioxidant activity of macromolecular bound active ingredients is far lower than that of their small molecule degradation products and free aglycone forms. Traditional processing products have limited free radical scavenging capabilities, and the efficacy of products is highly homogenized, making it difficult to meet the growing demand of the high-end health food market.

[0007] In response to the problems with the aforementioned technologies, some improved technical solutions have been disclosed in recent years: CN103704691A discloses a natto, propolis, and ginkgo leaf health food product and its preparation method, which involves simply physically mixing natto powder, ginkgo leaf extract, and propolis ethanol extract in a certain proportion. While this method enriches the product's efficacy to some extent, it merely uses physical mixing to simply combine the raw materials without any enzymatic hydrolysis or transformation of the macromolecular active ingredients in propolis and ginkgo leaves. The large molecular proteins in propolis and the glycoside-bound flavonoids in ginkgo leaves remain in a macromolecular bound state, making them difficult for the human body to directly absorb and utilize, thus failing to address the problem of low bioavailability. Furthermore, the product contents are prone to stratification and precipitation, resulting in poor formulation stability. More importantly, this method adds natto powder as a raw material, altering the original ingredient list and increasing the difficulty of formula compliance review and filing costs, which is detrimental to the rapid marketization of the product and the upgrading and iteration of existing products.

[0008] Existing technologies have attempted to process propolis using enzymatic hydrolysis. For example, CN111073942A discloses a propolis protein peptide and its preparation method, which processes propolis powder through microbial fermentation combined with protease hydrolysis to obtain propolis protein peptides. However, this method only targets the protein peptide processing of propolis as a single raw material and does not involve the simultaneous processing of ginkgo leaf components; its process involves microbial fermentation and high-temperature sterilization at 110~130℃, and high-temperature conditions can easily destroy heat-sensitive active components such as flavonoids and terpenes in propolis; the fermentation process has a long cycle, is difficult to control, and has low efficiency for industrial production. Furthermore, while the article "A Study on Enzymatic Hydrolysis and Antioxidant Activity of Flavonoid Glycosides in Propolis" (Bee Journal, 2007, Issue 1) theoretically explored the technical feasibility of enzymatic hydrolysis of flavonoid glycosides in propolis, pointing out that the antioxidant activity of flavonoid glycosides is far lower than that of flavonoid aglycones, and that enzymatic degradation of flavonoid glycosides can significantly enhance the antioxidant activity of propolis, this literature only remained at the theoretical discussion level. It did not provide specific and feasible enzymatic hydrolysis process schemes, nor did it address the peptide degradation of macromolecular proteins in propolis or the cell wall disruption of ginkgo leaves. None of the aforementioned individual enzymatic hydrolysis schemes can simultaneously achieve the three major objectives of propolis peptide conversion, ginkgo cell wall disruption, and flavonoid glycoside conversion within the same process system, resulting in limited synergistic effects and low process integration.

[0009] Some methods also employ acid, alkali, organic solvents, or harsh conditions to treat the raw materials. For example, CN108992473A discloses a method for extracting ginkgo leaf flavonoids, using steps such as lactic acid bacteria fermentation, ultrafine grinding, compound enzymatic hydrolysis (cellulase + pectinase), and flash extraction. However, in the compound enzymatic hydrolysis step, it uses a 40-75% ethanol solution as the enzymatic hydrolysis solvent and uses hydrochloric acid to adjust the pH to 4.2-4.6. The use of organic solvents and acids / alkalis poses risks of solvent residue and destruction of active ingredients. Another example is CN119097649A, which discloses a ginkgo leaf extract, preparation, and application for lowering blood sugar and lipids. It uses a biphasic enzymatic hydrolysis system for extraction, adding cellulase and β-glucosidase, as well as ethyl acetate and K2HPO4 buffer solution to the primary extract for enzymatic hydrolysis. This method also uses the organic solvent ethyl acetate and buffer salt system, which raises issues of solvent residue and desalting, increasing process complexity and food safety risks. Furthermore, it alters the product's "natural" and "chemical-free" attributes, contradicting the development trend of clean production and green processing. The acid, alkali, organic solvent or high temperature conditions in the above scheme can easily cause the heat-sensitive active ingredients such as flavonoids and terpene lactones to degrade, isomerize or oxidize, which not only reduces the bioactivity of the product, but may also introduce safety risks.

[0010] Some of the improved solutions require the introduction of high-end equipment such as supercritical extraction, membrane separation, and column chromatography, or involve complex processes such as microbial fermentation and flash extraction. The process routes are long, the investment costs are high, and the operation is difficult, making it difficult to directly promote and apply them on existing production lines and hindering industrialization.

[0011] The aforementioned disclosed prior art represents the current state of technology in this field regarding single raw material processing (propolis only or ginkgo leaves only), the use of organic solvents / acid-base reagents, simple physical mixing of new raw materials, and theoretical discussions without implementation schemes. None of these technologies have solved the technical challenges of simultaneously achieving propolis macromolecular protein peptide conversion, ginkgo leaf cell wall disruption, and flavonoid glycoside deglycosylation conversion in the same mild system. Furthermore, they have failed to achieve the compliant and synergistic goals of not adding new raw materials, not changing the ingredient list, and without chemical modification.

[0012] Therefore, there has long been a lack of a green preparation method in this field that does not require additional raw materials, does not change the ingredient list, is mild and efficient, simultaneously increases the content of small molecule peptides and free flavonoids, and can be directly adapted to existing production lines. This technological gap has become a key bottleneck restricting the quality upgrade and industrialization of propolis and ginkgo leaf compound products, and urgently needs to be overcome by those skilled in the art. Summary of the Invention

[0013] To address the technical problems of poor absorption, low stability, and raw material waste in traditional processes, as well as the issues of ingredient changes, activity degradation, and process incompatibility in existing improved solutions, the present invention aims to provide a method for preparing propolis-ginkgo leaf compound small molecule peptide enhancement. This method simultaneously enhances the peptide formation of propolis macromolecules, breaks down ginkgo cell walls, and efficiently converts flavonoid glycosides through a three-enzyme synergistic directional enzymatic hydrolysis system, significantly increasing the content of small molecule peptides and free flavonoids. It employs neutral, low-temperature, and mild conditions to maximize the preservation of flavonoid and terpene lactone activity, improving the product's antioxidant capacity and bioavailability. It optimizes product solubility and dispersibility, solves the problems of soft capsule layering and precipitation, and improves formulation stability. The process is simple, has low modification costs, and can be directly adapted to existing production lines, significantly improving raw material utilization and economic benefits. It is particularly suitable for enhancing the efficacy of processing without adding new raw materials or changing the ingredient list.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing small molecule peptides for enhancing the efficacy of propolis-ginkgo leaf composites involves mixing propolis extract and ginkgo leaf extract at a mass ratio of (80~90):(40~50) as raw materials, and then performing a synergistic enzymatic hydrolysis system of neutral protease, cellulase and glycosidase in a pure water system. The mixture is then inactivated, purified, concentrated and dried to produce a composite peptide powder. Specifically, the steps include the following: S1. Raw material mixing and dispersion: Select propolis extract and ginkgo leaf extract and mix them according to the corresponding mass ratio. Add 8 to 10 times the total mass of the raw materials of purified water and stir at 50 to 60°C for 20 to 40 minutes until fully wetted and dispersed. Filter through an 80 to 120 mesh filter to remove impurities and obtain a mixed dispersion. S2, Compound Targeted Enzymatic Hydrolysis: Adjust the pH of the mixed dispersion to 6.5-7.2, and add a compound enzyme preparation of neutral protease, cellulase and glycosidase; Based on the total dry matter weight of propolis extract and ginkgo leaf extract, the amount of neutral protease added is 0.8-1.2% of the dry weight of the material, the amount of cellulase added is 0.5-0.8% of the dry weight of the material, and the amount of glycosidase added is 0.3-0.5% of the dry weight of the material. The enzymatic hydrolysate is obtained by constant temperature stirring at 50-55℃ for 4-6 hours. S3, Inactivation and Refining: The enzyme hydrolysate is heated to 90-95℃ and kept at that temperature for 10-20 min to inactivate the enzyme. It is then allowed to stand at room temperature for 1-3 h to settle. The solution is then filtered through 150-250 mesh and 250-350 mesh respectively, and then centrifuged at 3000-4000 r / min for 10-20 min. The supernatant is then collected to obtain the purified active solution. S4. Low-temperature concentration and drying: The refined active liquid was vacuum concentrated to a solid content of 35-40% under vacuum conditions of 0.07-0.10 MPa and temperature ≤60℃, and then spray-dried at an inlet air temperature of 150-170℃ and an outlet air temperature of 75-85℃. The powder was then passed through a 60-100 mesh sieve to obtain propolis-ginkgo composite peptide powder. S5, Finished Product Compounding: The propolis-ginkgo composite peptide powder is mixed with food-grade excipients and homogenized by high-speed shearing for 30-50 minutes to complete the process.

[0015] In the above-mentioned raw material mixing and dispersion, purified water was used as the sole solvent, without the addition of any organic solvents or acid-base regulators. Constant temperature stirring promoted thorough wetting and uniform dispersion of the two extracts, and also provided a suitable temperature base for subsequent enzymatic hydrolysis. Neutral protease targeted and hydrolyzed the peptide bonds of the propolis macromolecular protein, degrading it into small peptide molecules that can be directly absorbed by the intestines; cellulase targeted and degraded the cellulose backbone in the cell wall of ginkgo leaves, breaking down the cell wall barrier and promoting the full release of intracellular active ingredients; glycosidase targeted and hydrolyzed the glycosidic bonds of flavonoid glycosides, converting bound flavonoids into free flavonoid aglycones. The effects of these three enzymes are not simply a functional additive, but rather form a synergistic relationship: cellulase's degradation of the cell wall creates a larger interaction interface and higher substrate accessibility for neutral protease and glycosidase; neutral protease's degradation of the protein improves the rheological properties of the system, promoting uniform contact between the enzyme and the substrate; and glycosidase's hydrolysis of glycosidic bonds removes the glycosyl modifications on the flavonoid molecules, exposing more active sites. The simultaneous action of these three components within the same mild system achieves a synergistic effect of "1+1+1>3". A four-stage purification process—inactivation, static sedimentation, staged filtration, and centrifugation—ensures that insoluble impurities in the enzymatic hydrolysis system are efficiently removed through physical means without the use of any chemical reagents, yielding a high-purity solution of the active ingredient.

[0016] Preferably, in S1, the total flavonoid content of the propolis extract is ≥30%; The total flavonoid content of the Ginkgo biloba leaf extract is ≥24%, the terpene lactone content is ≥6%, and the ginkgolic acid content is <5ppm; The mass ratio of the propolis extract to the ginkgo leaf extract is 85:45; The amount of purified water added is 9 times the total mass of the raw materials; the temperature of the constant temperature stirring is 55℃; the stirring time is 30 minutes; and the filter screen is 100 mesh.

[0017] Preferably, in S2, the neutral protease has an enzyme activity of 6000~12000 U / g, the cellulase has an enzyme activity of 6000~12000 U / g, and the glycosidase has an enzyme activity of 3000~7000 U / g. The neutral protease, cellulase, and glycosidase mentioned are all food-grade enzyme preparations. The three enzymes form a synergistic and directional enzymatic hydrolysis system under conditions of pH 6.5~7.2 and 50~55℃.

[0018] Preferably, the neutral protease has an enzyme activity of 10,000 U / g, the cellulase has an enzyme activity of 8,000 U / g, and the glycosidase has an enzyme activity of 5,000 U / g.

[0019] Preferably, in step S2, based on the total dry matter weight of propolis extract and ginkgo leaf extract, the amount of neutral protease added is 1.0% of the dry weight of the material, the amount of cellulase added is 0.6% of the dry weight of the material, and the amount of glycosidase added is 0.4% of the dry weight of the material. The enzymatic hydrolysis was performed at a pH of 6.8, a temperature of 52°C, and a time of 5 hours.

[0020] Preferably, in step S3, the inactivation temperature is 92°C, the inactivation time is 15 min, the settling time is 2 h, the material is filtered through 200 mesh and 300 mesh filters in sequence, the centrifugation speed is 3500 r / min, and the centrifugation time is 15 min.

[0021] Preferably, in step S4, the vacuum concentration has a vacuum degree of 0.08~0.09 MPa, a temperature of ≤60℃, and a solid content of 38%. The inlet air temperature for spray drying is 160℃, and the outlet air temperature is 80℃.

[0022] Preferably, in step S5, the content of small molecule peptides in the propolis-ginkgo composite peptide powder is 18.5%~20.0%, and the proportion of free flavonoids to total flavonoids is 82~85%. The food-grade excipient is a mixture of PEG400 and medium-chain triglycerides; The mass ratio of the propolis-ginkgo complex peptide powder to PEG400 and medium-chain triglycerides is (30~40):(35~45):(20~30).

[0023] A propolis-ginkgo leaf composite small molecule peptide product is obtained by the preparation method described above.

[0024] Preferably, the product includes any one of soft capsules, compressed candies, solid beverages, or oral liquids.

[0025] Compared with the prior art, the present invention has at least the following technical effects: This invention provides a method for preparing a propolis-ginkgo leaf compound with small molecule peptide enhancement. This method utilizes a three-enzyme synergistic targeted enzymatic hydrolysis system to simultaneously enhance the peptide formation of propolis macromolecules, break down ginkgo cell walls, and efficiently convert flavonoid glycosides, significantly increasing the content of small molecule peptides and free flavonoids. Neutral, low-temperature, and mild conditions are employed to maximize the preservation of flavonoid and terpene lactone activity, improving the product's antioxidant capacity and bioavailability. The method optimizes product solubility and dispersibility, resolving soft capsule layering and precipitation issues, and improving formulation stability. The process is simple, has low modification costs, and can be directly adapted to existing production lines, significantly improving raw material utilization and economic benefits. It is particularly suitable for enhancing efficacy without adding new raw materials or changing the ingredient list.

[0026] This preparation method has the following innovations: (1) Three-enzyme synergistic directional catalysis innovation: For the first time, neutral protease + cellulase + glycosidase are combined in a specific ratio to form a synergistic system that targets proteins, cell walls and flavonoid glycosides respectively, and completes the three major transformations simultaneously in the same neutral low temperature system, which is not a simple enzyme superposition; (2) Zero-additive compliant innovation: No new raw materials, no changes to the ingredient list, no acid / base / organic solvent modification, only through biological enzymatic hydrolysis and physical purification to achieve significant efficiency improvement, solving the problems of formula compliance and filing; (3) Mild and efficient conversion innovation: Neutral low temperature conditions avoid activity destruction, small molecule peptide content ≥18.5%, free flavonoids ≥82%, absorption efficiency is increased by more than 3 times, and antioxidant capacity is significantly enhanced; (4) Process compatibility and mass production innovation: It can be directly connected to existing production lines without large-scale modification, and the raw material utilization rate is ≥91%, solving the problem of industrialization. (5) Innovative formulation stability: Improved solubility and dispersibility, soft capsules do not separate or precipitate, and shelf-life stability is greatly improved.

[0027] The absorption efficiency of this propolis-ginkgo leaf compound small molecule peptide product is significantly improved. In synergy with free flavonoids, the DPPH clearance rate is increased by 82.22%. The solubility and dispersibility are improved, the contents of the soft capsules are uniform and stable, and the shelf life is extended. It degrades macromolecular sensitizing impurities and reduces the risk of adverse reactions. The raw material utilization rate is increased from 58% to ≥91%, the process is adapted to existing production lines, and the production cost is reduced. No new raw materials are added and the ingredient list is not changed, which reduces the risks of filing and market promotion. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the overall process flow of the preparation method in the specific implementation embodiment; Figure 2This is a schematic diagram illustrating the structural changes of the complex enzymatic hydrolysis peptides during the preparation process of Example 1. Figure 3 A bar chart comparing the active ingredients of products prepared by conventional processes and those prepared according to Example 1 of this invention; Figure 4 This is a comparison chart of the product's microstructure and solubility / dispersibility. Detailed Implementation

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0030] One specific embodiment of the present invention is as follows: The process of this invention, through adjusting the amount of enzyme added, the enzymatic hydrolysis time, and the pH setting, forms three typical embodiments, all of which fall within the scope of protection of the claims, and the effects are stable and consistent.

[0031] A method for preparing a propolis-ginkgo leaf compound with small molecule peptides for enhanced efficacy includes the following steps: (See schematic diagram of the overall process below.) Figure 1 As shown: 1. Raw material acceptance and pretreatment: 1.1. Raw Material Acceptance Standards 1.1.1 Acceptance Criteria for Propolis Extract 1) Appearance: Fine powder ranging from brownish-yellow to brownish-red, free of lumps, mold spots, and odor; 2) Core indicators: Total flavonoid content ≥30% (detected by ultraviolet spectrophotometry); Moisture ≤6.0%; Ash ≤8.0%; Heavy metals (lead ≤2.0mg / kg, arsenic ≤1.0mg / kg, mercury ≤0.3mg / kg) meet the national standards for health food raw materials; Microbial limits: Total bacterial count ≤1000CFU / g, free of Salmonella and Staphylococcus aureus.

[0032] 3) Qualification requirements: Suppliers must provide raw material quality inspection reports, food-grade production qualifications, and batch traceability documents.

[0033] 1.1.2 Acceptance Criteria for Ginkgo Biloba Extract 1) Appearance: Light yellow to yellowish-brown free-flowing powder, free of impurities and rancid odor; 2) Core indicators: Total flavonoids ≥24%, terpene lactones ≥6%, ginkgolic acid content <5ppm (detected by high performance liquid chromatography); moisture ≤5.0%; heavy metals same as those in propolis extract; 3) Microbial limits: Molds and yeasts ≤100 CFU / g, no pathogenic bacteria; 4) Qualifications: Includes a special test report on deginkgolic acid and a food-grade extract registration certificate.

[0034] 1.1.3 Acceptance of excipients / enzyme preparations Neutral protease, cellulase, and glycosidase are all food-grade, and corresponding enzyme activity test reports are provided; the soft capsule excipients PEG400 and medium-chain triglycerides (MCT oil) meet the national standards for food additives.

[0035] 1.2. Raw material pretreatment process 1) Sorting and sieving to remove impurities: The qualified propolis extract and ginkgo leaf extract are sieved through a 60-mesh standard sieve to remove foreign impurities such as lumps, hair, and debris; the lumpy raw materials are pulverized at a low temperature of 40℃ and then sieved twice to ensure that the powder is fine and free of coarse lumps.

[0036] 2) Temperature and humidity balancing conditioning: The two types of raw materials after screening are transferred to a closed constant temperature and humidity silo. The temperature inside the silo is controlled at 20~25℃ and the relative humidity at 45%~55%. The materials are left to stand for 24 hours to balance and eliminate moisture fluctuations between different batches of raw materials, so as to prevent clumping and uneven dissolution when water is added later.

[0037] 3) Premixing and metering: According to the production ratio of 85:45, use a high-precision electronic scale to weigh out the propolis extract and ginkgo leaf extract separately, and put them into a closed mixing tank for low-speed stirring and premixing for 5 minutes to obtain a uniform premixed raw material powder. After pretreatment, the raw material is sent to the next raw material mixing and dispersion process.

[0038] 1.3 Control of Non-conforming Raw Materials Raw materials that fail to meet one or more of the required standards should be stored separately, labeled as non-compliant, and returned to the upstream supplier. They must not be used in the production line. Samples for re-inspection should be retained for at least 6 months.

[0039] 2. Raw material mixing and dispersion: Propolis extract (total flavonoids ≥30%) and Ginkgo biloba extract (total flavonoids ≥24%, terpene lactones ≥6%, ginkgolic acid <5ppm) are mixed at a mass ratio of 85:45, 8~10 times the amount of purified water is added, the mixture is stirred at 55℃ for 30min, and filtered through 100 mesh to remove macromolecular impurities to obtain a mixed dispersion. 3. Compound targeted enzymatic hydrolysis: Adjust the pH to 6.5~7.2, add 0.8%~1.2% neutral protease, 0.5%~0.8% cellulase, and 0.3%~0.5% glycosidase, and enzymatically hydrolyze at 50~55℃ for 4~6 hours to achieve simultaneous synergistic effects on multiple targets; 4. Enzyme inactivation + purification: Incubate at 90~95℃ for 15 min for inactivation, let stand for 2 h, filter through 200 mesh → 300 mesh, centrifuge at 3500 r / min for 15 min to obtain supernatant purified active solution; 5. Low-temperature concentration and drying: Vacuum concentration at 0.08~0.09MPa and ≤60℃ until the solid content is 35%~40%, spray drying at 160℃ inlet and 80℃ outlet, and sieve through 80 mesh to obtain composite peptide powder; 6. Finished product compounding: The compound peptide powder and conventional excipients are homogenized by high-speed shearing for 40 minutes to form soft capsules; 7. Finished product inspection and warehousing. Supporting finished product inspection standards: Testing indicators include five items: small molecule peptide content, free flavonoid ratio, DPPH free radical scavenging rate, raw material utilization rate, and formulation stratification and precipitation stability. Each batch of finished products can only be warehoused after all tests are passed.

[0040] Example 1: A method for preparing a propolis-ginkgo leaf compound with small molecule peptides for enhanced efficacy includes the following steps: Weigh out 85 parts of propolis extract and 45 parts of ginkgo leaf extract, mix them, add 9 times the amount of purified water, stir and disperse at 55℃ for 30 min, and filter through 100 mesh; adjust the pH to 6.8, add 1.0% neutral protease, 0.6% cellulase and 0.4% glycosidase, and enzymatically hydrolyze at 52℃ for 5 h; inactivate at 92℃ for 15 min, filter through 200 mesh → 300 mesh, centrifuge at 3500 r / min for 15 min; vacuum concentrate to 38% solids, spray dry (inlet air 160℃ / outlet air 80℃) to obtain composite peptide powder; mix the composite peptide powder with PEG400 and MCT oil at a mass ratio of 35:40:25 and homogenize to prepare soft capsules.

[0041] Example 2: Low-enzyme-volume, short-time preparation method Weigh out 85 parts of propolis extract and 45 parts of ginkgo leaf extract, mix them, add 8 times the amount of purified water, stir and disperse at 55℃ for 30 min, and filter through 100 mesh; adjust the pH to 6.5, add 0.8% neutral protease, 0.5% cellulase and 0.3% glycosidase, and enzymatically hydrolyze at 50℃ for 4 h; inactivate at 90℃ for 15 min, filter through 200 mesh → 300 mesh, centrifuge at 3500 r / min for 15 min; vacuum concentrate to 35% solids, spray dry (inlet air 160℃ / outlet air 80℃) to obtain composite peptide powder; mix the composite peptide powder with PEG400 and MCT oil at a mass ratio of 30:45:25 and homogenize to prepare soft capsules.

[0042] Example 3: High-enzyme-volume, long-term preparation method Weigh out 85 parts of propolis extract and 45 parts of ginkgo leaf extract, mix them, add 10 times the amount of purified water, stir and disperse at 55℃ for 30 min, and filter through 100 mesh; adjust the pH to 7.2, add 1.2% neutral protease, 0.8% cellulase, and 0.5% glycosidase, and enzymatically hydrolyze at 55℃ for 6 h; inactivate at 95℃ for 15 min, filter through 200 mesh → 300 mesh, centrifuge at 3500 r / min for 15 min; vacuum concentrate to 40% solids, spray dry (inlet air 160℃ / outlet air 80℃) to obtain composite peptide powder; mix the composite peptide powder with PEG400 and MCT oil at a mass ratio of 40:38:22 and homogenize to prepare soft capsules.

[0043] Comparative example: Complete process steps and parameters for traditional ethanol extraction: Take 85 parts of propolis extract and 45 parts of ginkgo leaf extract with the same ratio as in the example, add 10 times the total mass of raw materials in 70% ethanol aqueous solution, and extract twice at a constant temperature of 70℃, with each extraction lasting 2 hours; combine the two extraction filtrates, recover ethanol under reduced pressure, dry in a vacuum drying oven at 65℃ until the moisture content is ≤5%, and pulverize through an 80-mesh sieve to obtain traditional composite powder; take an equal amount of powder and compound it with PEG400 and MCT oil excipients that are exactly the same as in Example 1, homogenize, and prepare soft capsules. The entire process is without enzymatic hydrolysis and peptide conversion treatment, and the remaining post-processing and formulation conditions are consistent with the three sets of examples.

[0044] The performance comparison is shown in Table 1 below:

[0045] The above data shows that the three sets of embodiments of the present invention are significantly superior to the traditional alcohol extraction control in all key performance indicators, proving the effectiveness and superiority of the technical solution of the present invention. Among them, Embodiment 1 is the optimal implementation scheme.

[0046] like Figure 2The diagram shows the structural changes of peptides synthesized by enzymatic hydrolysis. On the left: the traditional process produces a macromolecular bound state with a coarse network structure linked by glycosidic bonds. This macromolecular bound state is poorly absorbed. On the right: the small molecule peptide + free flavonoid monomer of Example 1 of this invention has a short-chain, fine-line structure. This small molecule peptide + free flavonoid structure is easily absorbed.

[0047] The comparison results show that the product of Example 1 of the present invention represents a fundamental transformation at the molecular level.

[0048] like Figure 3 The chart shown is a bar chart comparing the active ingredients of the products produced by the traditional process and those produced by Example 1 of the present invention. The horizontal axis of the chart contains three sets of comparative data: small molecule peptide content, free flavonoid ratio, and raw material utilization rate. Gray bars represent the traditional process, and black bars represent Example 1 of the present invention.

[0049] Figure 3 The data results are readily apparent: the content of small molecule peptides, the proportion of free flavonoids, and the utilization rate of raw materials in this invention are all superior to those of traditional processes, with the utilization rate of raw materials increasing by more than 33 percentage points. The bar chart does not show the DPPH free radical scavenging rate data. This antioxidant indicator is an in vitro activity detection item, which is determined independently by spectrophotometry and is not reflected in the bar chart of this component content.

[0050] like Figure 4 The image shows a comparison of the product's microstructure and solubility / dispersibility, divided into two sections, a and b. Section A compares the microscopic particle morphology: On the left, traditional processes are labeled "granules, coarse particles, large agglomerations," indicating that traditional powder particles are large and severely agglomerated; on the right, the present invention is labeled "fine and uniform," indicating that molecular degradation after enzymatic hydrolysis refines the powder particles and makes them more evenly dispersed. Part B is a comparison of the dispersion stability of aqueous solutions: The three markings in the traditional process area on the left represent: ① the upper clear liquid area, ② the middle black precipitate layer, and ③ the bottom fine sedimented particles, which together form a stratified precipitation phenomenon; the area of ​​the present invention on the right shows no stratification or precipitation, and is clear and uniform overall, indicating a significant improvement in water solubility and dispersion stability.

[0051] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing small molecule peptide-enhanced propolis-ginkgo leaf composites, characterized in that, Using a mixture of propolis extract and ginkgo leaf extract at a mass ratio of (80~90):(40~50) as raw materials, a three-enzyme synergistic directional enzymatic hydrolysis system of neutral protease, cellulase and glycosidase is carried out in a pure water system; then, the mixture is inactivated, purified, concentrated and dried to produce a complex peptide powder. Specifically, the steps include the following: S1. Raw material mixing and dispersion: Select propolis extract and ginkgo leaf extract and mix them according to the corresponding mass ratio. Add 8 to 10 times the total mass of the raw materials of purified water and stir at 50 to 60°C for 20 to 40 minutes until fully wetted and dispersed. Filter through an 80 to 120 mesh filter to remove impurities and obtain a mixed dispersion. S2, Compound Targeted Enzymatic Hydrolysis: Adjust the pH of the mixed dispersion to 6.5-7.2, and add a compound enzyme preparation of neutral protease, cellulase and glycosidase; Based on the total dry matter weight of propolis extract and ginkgo leaf extract, the amount of neutral protease added is 0.8-1.2% of the dry weight of the material, the amount of cellulase added is 0.5-0.8% of the dry weight of the material, and the amount of glycosidase added is 0.3-0.5% of the dry weight of the material. The enzymatic hydrolysate is obtained by constant temperature stirring at 50-55℃ for 4-6 hours. S3, Inactivation and Refining: The enzyme hydrolysate is heated to 90-95℃ and kept at that temperature for 10-20 min to inactivate the enzyme. It is then allowed to stand at room temperature for 1-3 h to settle. The solution is then filtered through 150-250 mesh and 250-350 mesh respectively, and then centrifuged at 3000-4000 r / min for 10-20 min. The supernatant is then collected to obtain the purified active solution. S4. Low-temperature concentration and drying: The refined active liquid was vacuum concentrated to a solid content of 35-40% under vacuum conditions of 0.07-0.10 MPa and temperature ≤60℃, and then spray-dried at an inlet air temperature of 150-170℃ and an outlet air temperature of 75-85℃. The powder was then passed through a 60-100 mesh sieve to obtain propolis-ginkgo composite peptide powder. S5, Finished Product Compounding: The propolis-ginkgo composite peptide powder is mixed with food-grade excipients and homogenized by high-speed shearing for 30-50 minutes to complete the process.

2. The preparation method according to claim 1, characterized in that, In S1, the total flavonoid content of the propolis extract is ≥30%; The total flavonoid content of the Ginkgo biloba leaf extract is ≥24%, the terpene lactone content is ≥6%, and the ginkgolic acid content is <5ppm; The mass ratio of the propolis extract to the ginkgo leaf extract is 85:45; The amount of purified water added is 9 times the total mass of the raw materials; the temperature of the constant temperature stirring is 55℃; the stirring time is 30 minutes; and the filter screen is 100 mesh.

3. The preparation method according to claim 1, characterized in that, In S2, the activity of the neutral protease is 6000~12000 U / g, the activity of the cellulase is 6000~12000 U / g, and the activity of the glycosidase is 3000~7000 U / g. The neutral protease, cellulase, and glycosidase mentioned are all food-grade enzyme preparations. The three enzymes form a synergistic and directional enzymatic hydrolysis system under conditions of pH 6.5~7.2 and 50~55℃.

4. The preparation method according to claim 3, characterized in that, The neutral protease has an enzyme activity of 10,000 U / g, the cellulase has an enzyme activity of 8,000 U / g, and the glycosidase has an enzyme activity of 5,000 U / g.

5. The preparation method according to claim 1, characterized in that, In step S2, based on the total dry matter mass of propolis extract and ginkgo leaf extract, the amount of neutral protease added is 1.0% of the dry weight of the material, the amount of cellulase added is 0.6% of the dry weight of the material, and the amount of glycosidase added is 0.4% of the dry weight of the material. The enzymatic hydrolysis was performed at a pH of 6.8, a temperature of 52°C, and a time of 5 hours.

6. The preparation method according to claim 1, characterized in that, In step S3, the inactivation temperature is 92℃, the inactivation time is 15min, the settling time is 2h, and the material is filtered through 200 mesh and 300 mesh fine filters in sequence. The centrifugation speed is 3500r / min, and the centrifugation time is 15min.

7. The preparation method according to claim 1, characterized in that, In step S4, the vacuum concentration has a vacuum degree of 0.08~0.09 MPa, a temperature of ≤60℃, and a solid content of 38%. The inlet air temperature for spray drying is 160℃, and the outlet air temperature is 80℃.

8. The preparation method according to claim 1, characterized in that, In S5, the content of small molecule peptides in the propolis-ginkgo composite peptide powder is 18.5%~20.0%, and the proportion of free flavonoids to total flavonoids is 82~85%. The food-grade excipient is a mixture of PEG400 and medium-chain triglycerides; The mass ratio of the propolis-ginkgo complex peptide powder to PEG400 and medium-chain triglycerides is (30~40):(35~45):(20~30).

9. A propolis-ginkgo leaf composite small molecule peptide product, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 8.

10. A propolis-ginkgo leaf composite small molecule peptide product according to claim 9, characterized in that, The product includes any one of soft capsules, compressed candies, solid beverages, or oral liquids.

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